Art of butyl-acetonic fermentation



Patented May s, 1928.

UNITED STATES PATENT OFFICE.

DAVID A LEGG, OF TERIQE HAUTE, INDIANA,

ASSIGNOR '1'0 COMMERCIAL SOLVENT.

CORPORATION, or rnn'nn naurn, INDIANA, A coaronn'non or mum.

ART OF BUTYL-ACETONIC FERMENTATION.

No Drawing.

- 1n the presence of adverse fermentation conditions.

The butyl-acetonie fermentation of carbohydrates has been carried out on a large manufacturing scale for some years past.

In this process a sterile mash of amylaceous or saccharine carbohydrates is prepared and the warm mash is inoculated with a culture of b'utyl-acetonic bacilli, whereupon fermentation proceeds at a temperature of about 36 0., this being the optimum fermentation temperature maintained by the bacilli with strains have yield represents the .total butylicum,

'bohydrates, a solv 'cohol (about two parts) out the outside application of heat.

Inthe case of sterile corn mash'of 8% concentratiom'a normal fermentation is com-' plete within thirty-six to seventy-two hours and there is a solvent yield of about 25% by Weight of the dry corn. This solvent quantity of butyl alcohol, acetone, and ethyl alcohol obtained. The fermented mash is then distilled in the ordinary manner to rempve the solvents present in the aqueous solution.

Butyl-ace'tonic bacilli are present in nature and may be isolated and purified according to the method of Weizmann (U. S. Patent 1,315,585). cillus has previously been described by various names, including :Bacillus granulebacter pectinovorum, Bacillus amylobacter, Clostridium butyricum, Bacillus butyricus, Bacillus butyl-aceticum, Bacillus aceto- Clostridiuln aceto-butylicum, etc. Sli ht difierences due to culturing methods and treatment have been observed but all certain definatory characteristics. y

The butyl-acetonic bacillus is chiefly characterized by its ability to produce, from carent mixture of butyl alacetone (about one part) and, usually, small quantities of ethyl alcohol. A mixture of hydrogen gas and carbon dioxide gas is given ofl during fermentation. During the first stages of fermentation the mash acidity rapidly increases to a peak of about 46'c. c. of N/lO sodium hydroxide per 10 c. c. of mash. Later, during fermentation, the mash acidity Application filed October 1, 1926. Serial Ho. 139,016.

The butyl-acetonic ba eElssuEn is lesseneduntil, in a normal ca-se it is about 2-3 c. 0. when, fermentation ceases. The

butyl-a'cetonic bacillus, further, has the power to operate on starch directly, and does not require a preliminary conversion of this material to sugar.

- The bacillus is an anaerobe but is ordinarily employed under practical aerobic conditions. Young vegetative cells stain yellow with iodine, but later, as sporulation approaches, the cells take on clostridial form, whereupon they stain blue or violet with iodine owing to the presence of granulose.

Young vegetative cells are actively motile with from nine to eighteen peritrichous flagella. They are at firstgram-positive, but after eighteen to twenty-four hours shrivelled gram-negative cells increase in numbers.

lVhile a pure culture of butyl-acetonic bacilli will produce a factory solvent yield of 25% based on dry corn, or a similar figure when other carbohydrates are fermented, the history of the art has shown recurrent periods of lowered yields. due to abnormal fermentation. There 'are known in the art two distinctly different ty es of abnormal fermentation which-are ordinarily classified as contaminated fermentation and sluggish fermentation.

Contaminated fermentation, as the name implies, is due, to the presence in the fermenting mash of other organisms alon with the butyl-acetonic bacilli. Lactic aci producing organisms such as B. Volutans,. once present in the mash, attack the carbo--. hydrate present with great activity and may diminishor almost entirely preclude the normal fermentation. The prevention of contamination in the butyl-aeetonic fermentation art has been the subject of much study and effort, and those skilled in the art are now well able to prevent the appearance of contaminating organisms in the fermentation by proper precautions in sterilization and operation; and, if contaminating organisms do find their way into the mash, it

no apparent reasons. It is usually epidemic in character and of sudden incidence.

When sluggish fermentation is once noticed in one or more of the vessels employed in a butyl-acetonic fermentation plant within about twenty four hours it will frequently become apparent in hundreds of disconnected vessels of various sizes, including laboratory cultures. ,Even cultures which have been stored for years in sealed tubes in spore form, when transferred to and gown in a sterile mash are not immune om sluggishness during a plant epidemic period. I

Sluggish fermentation is primarily characterized by a prolonged acidity peak and by the extreme slowness with which the end point is reached. An epidemic of sluggishness greatly reduces the total solvents producible from'apparatus of a given size and standard capacity in a stated time. During such an epidemic, production schedules are overthrown and great loss ensues.

Fully as important as the factor of slow-- ness which occurs in sluggish fermentation, is the factor of diminished yield of solvents. Sluggish fermentations are rarely complete fermentations-all of the fermentable carbohydrate is not fermented, and an appreciable loss ensues, both in reduced solvent yield and in the increased time required for fermentation.

I have now discovered a means of operating the butyl-acetonic fermentationprocess in a manner that prevents the loss of time and loss of solvents otherwise incurred through sluggishness, and a process which 'The recurrent and epidemic character of 'ves uniformly higher plant yields than t ose known in the art.

My invention is best understood in connection with my theory for its operativeness though it is of course understood that my in-.-'

vention stands distinct from the .theory.

slug shness suggests that it is a disease of t e butyl-acetonic bacilli. The precise circumstances under which a fermentation process, operatin normally, first falls under the. influence of s uggishness are not known. It is certain, however, that when the mash in one fermenting vessel becomes afiected with sluggishness there is usually a sudden appearance of the disease in a great number 0 other vessels.

The slightest contact: of anyportion of a slu gish mash with another mash-is sufficient to induce sluggishness. If a sample of sluggish fermenting mash is filtered, first through a Buechner filter to remove all solids and then through a Berkefeld filter to remove bacteria, one drop of the filtrate placed in normal fermenting. mash is sufiicient to induce sluggishness. Indeed, if one drop of the filtrate is diluted one million times, a drop of the resultant diluted liquid 'mally fermenting mash.-

gishness in 11 nor-' likewise the sluggishnessmay be propagated indefinitely by transferring one drop of the Berkefel filtrate from a mash undergoin mentation'to a fresh mash, ermenting the fresh mash for about twenty four hours, se curing a Berke'feld filtrate from it, and repeating the cycle. Such transferring does not involve ness-whatever its cause-becomes as pro- Eounccd in the second mash trait was in the rst. On the basis of these facts, and on others will frequently induce slu not reported her.e, it would appear that sluggish fermentation is caused by a living organism of ultramicroscopic dimensions. If this is the case it may be either a saprophyte living in association with the butyl-acetonic organism, or it may be a true parasite or bacteriophage.

I have now discovered a process whereby butyl-acctonic bacilli may be rendered resistant to epidemics of sluggishness, whereby a normal yield of solvents is obtainable at all W times from carbohydrate fermentation without any interference from epidemics of sluggislmess, or, in the language of my theory, quite regardless of the presence of an ultrathe butyl-acetonic bacillus to the presence of the ultra-virus (according to my theory) or at any rate to whatever influencing agent is present in a sluggishly-fermenting mash or the Berkefeld filtrate therefrom.

I accomplish this immunization by repeatly subcultivating butyl-acetone bacilli in the presence of the filtrate from a sluggish fermentation, the cultures being heatshocked before each transfer.

More specifically, the immunization of the 1 butyl-acetonic bacillus may be accomplished as follows:

A sterile carbohydrate mash, consisting,

which there is added a small portion, one .or 1

more drops, of clear liquid obtained from a carbohydrate mash undergoing a sluggish butyl-acetonic fermentation by filtering the mash first to remove all solids and second through a Berkefeld filter to remove bac- 0.

teria. Fermentation is then allowed to proceed at a temperature of about 36 C. for four or more days-i. e.'until spores have developed. This spore culture is then used to inoculate fresh corn mash which is again heat-shocked for about three minutes at 100 C., andtreated with two drops of the filtrate as previously mentioned. The' second fermentation is allowed to proceed as before and additional transfers withshocksluggish fer- 7o true dilution, as the sluggishl5 bacteriophage 1n .the mash. 1' achieve this end by immunizing cultures of ing and with filtrate addition are madethat is, the process is repeated. At the end of about ten such treatments the culture thus obtained in spore form at the end of the last treatment will ordinarily be found to be immunized in respect to sluggish fermentation, and. if not, the described treatment of the culture is continued until immunity is attained. The extent of immunity is measured by inoculating a sterile mesh with the immune culture, adding a few drops of the Berkefeld filtrate of a sluggish fermentation, and allowing fermentation to proceed. A fermentation giving a normal solvent yield within a normal time indicates that the culture is completely immunized.

In making such tests, one employs, as controls, a normal-.non immunized culture for fermenting a sterile carbohydrate mash, the result being taken as an index of yield and of fermentation time, and one also employs a normal non-immunized culture for fermenting a sterile carbohydrate mash in the presence of filtrate from a sluggish fermentation,'the result being compared with theresult obtained from immunized cultures. The improved results attained with im- -munized cultures are illustrated by the following tabulation Average solvent yield of 4 flask ferplantations Number of immunizing treatments given culture Culture employed None. None. None.

v, In addition to clearly disclosing the bane-- ficial result attained by the use of immunized cultures the tabulation also is useful in showing that the process ofimmunization does not transform low-yielding cultures into high-yielding cultures. For example, Culture R 4, while immunized and thus capable of producing a normal fermentation in the presence of the ultra-virus, gives consistently lower results than its companion cultures. If high solvent yields are to be attained, the culture selected to be immunized must be a normal high-yielding culture under optimum fermentation conditions. Y J

It will also be noted that while the control (non-immunized) cultures shown inthe tabulation consistently give much lower This unistic of biological processes. The point of importance is that non-immunized cultures give lower average yields than immunized cultures.

lVhile in describing my process for immunizing cultures I have stated that cultures should be heat-shocked for three minutes at 100 (1, it should be understood that temperature and duration of heatshocking may be varied in accordance with well-known bacteriological technique. The purpose of heat-shocking is to eliminate vegetative forms of the bacillus, leaving only the spores to germinate. So long as this result is attained, the precise method of shocking may be varied.

When an immunized culture is added to a carbohydrate mash containing the filtrate from sluggish fermentation there is produced a normal, rapid, and com lete fermentation with a good solvent yie d. This, of course, is the effect observed when a laboratory test of the quality of the culture thus obtained is made. Practically, in manufacturing operations, the beneficial effect of the use of such a culture is rofound. sluggish fermentation as such, isappears from the factory.

While the ultra virus believed to cause sluggishness may still have recurrent periods of activity such that extreme sluggish fermentation would occur if ordina cultures were used, when immunized resistant) cultures are employed there is no period of sluggish fermentation actually ex rienced in manufacturingoperations, and t e butylacetonic fermentation process may be operated to consistently produce good yields of solvents.

A further very great advanta e accrues from the use of immunized resistant) butyl-a"etonic bacilli in conducting the butyl-acetonic fermentation processname- 1y that consistently higher yields of solvents are obtained at all times in the manufacturing operations, than are obtained when corresponding non-immunized cultures are used. For example, under normal plantconditions and using non-immunized cultures the solvent yield,based on dry corn has seldom been over 24%, when reckoned over an operating period of several months. However, when my immunized (resistant) cultures are employed, the solvent ield'un den plant" conditions is maintain consisteritly' at 25% or more,-=based on dry corn.

In this connection it should be noted that.

generally the practice of theart, solvent yields obtained in large scale manufacturmg operations are not as great as the best yields obtained in laboratory experiments.

There is probably a close connection between the ability of m immunized (resistant) cultures to pr uce normal solvent yields from mashes containing the ultravirus or bacteriophage causin sluggishness, and their ability to pro uce higher yields of solvents in plant practice at all times, whether or not sluggishness is apparent.

Thus, it is possible that in plant practice the ultra-virus causing sluggishness is almost always present and that actual epidemics of sluggishness are due to an increase of the virulence of the ultra-virus. At other times its virulence may be only sufficient to cause a slightlowering of the final solvent yield obtained from non-immunized cultures, without producing the characteristic symptoms of sluggish fermentation.

While I have described the process of immunization as bein referentially conducted with some Ber efiald filtrate from a sluggish fermentation, the filtrate from an other bacterial filter might be employe Althoughv any portion of a sluggishly-fermenting mash might be used to introduce the virus, the use of unfiltered mash is not referred, since such procedure will introuce strains of the Bacillus other than the one undergoin treatment, for the filtrate is not prdinarrly obtained from the culture being 1mmunized. ile I have described specifically the use of corn as a carbohydrate for fermentation it should be pointed 'out that any fermentable carboydrate saccharine or amylaceous is useful with'my immunized (resistant) cultures. In the appended claims, my im roved cultures are described as immunized and it should be understood that this term is used as syno'nymous'with the term resistant, as was heretofore indicated.

Now, having described my invention, 1 claim the following as new and novel 1. A process for the production of butyl alcohol and acetone which comprises preparing a sterile carbohydrate mash, and moon ating said mash with a culture of the hereinabove-described immunized butyl-acetomc bacillus. i

2. A process for the production of butyl alcohol and acetone which comprises inoculatlng a sterile carbohydrate mash with a culture of the hereinabove-described immunized butyl-acetonic bacillus, permitting the fermentation to go to completion, and

recovering butyl alcohol" and acetone fronr the fermented mash.

- 3. A process for the production of butyl alcohol and acetone which comprises pre paring a sterile amylaceous mash, and inoculating said mash with a culture of the hereinabove-described immunized butyl-acetonic bacillus.

4. A process for the production of-butyl alcohol and acetone which comprises inoculating a sterile amylaceous mash with a culture of the hereinahove-described immunized butyl-acetonic bacillus, permitting the fermentation to go to completion, and recovering butyl alcohol and acetone from the fermented mash.

5. A process for producting an immunized culture of butyl-acetone bacilli which comprises repeatedly sub-cultivating said bacilli in carbohydrate media in the presence of some sluggishly-fermenting carbohydrate mash, the cultures being heatshocked at each transfer attenuated vegetative cells.

6. A process for producing an immunized culture of butyl-acetonic bacilli which comprises re eatedly sub-cultivating said bacilli in car ohydrate media in the presence of some sluggishly-fermenting carbohydrate mash, the cultures being shocked for three minutes at 100 C. at the time of each transfer to eliminate attenuated vegetative cells.

7. A process for producing an immunized culture of butyl-acetonic bacilli which com rises re eatedly sub-cultivating said baci li in car ohydrate media in the presence of a portion of a Berkefeld filtrate of a carbohydrate mash which is undergoing sluggish butyl-acetonic fermentation, the cultures being heat-shocked at each transfer to eliminate attenuated vegetative cells.

8. A process for producing an immunized culture of butyl-acetonic'bacilli which comprises sub-cultivating said bacilli ten or more times in a carbohydrate medium in the presence of a portion of a Berkefeld filtrate of a carbohydrate mash which is undergoing sluggish butyl-acetonic fermentation, the cultures being heat-shocked at each transfor to eliminate attenuated vegetative cells.

In testimony whereof I alfix my signature.

DAVID A. LEGG.

to eliminate, 

